Peptide antagonists of vascular endothelial growth factor and methods of use thereof

ABSTRACT

The present invention provides isolated polypeptides having VEGF antagonist activity, pharmaceutical compositions and methods of treatment. The polypeptides of the invention include polypeptides comprising a portion of SEQ ID NO: 1 having VEGF antagonist activity, polypeptides comprising SEQ ID NO: 2 or a portion thereof having VEGF antagonist activity, and a polypeptide having the structure of formula (I), set forth above. The present invention further includes analogs and derivatives of these polypeptides having VEGF antagonist activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/172,491 filed on Jul. 14, 2008, which is a continuation of U.S.application Ser. No. 10/858,849 filed on Jun. 2, 2004, now U.S. Pat. No.7,414,027 issued on Aug. 19, 2008, which is a continuation of U.S.application Ser. No. 09/579,420 filed on May 25, 2000, now U.S. Pat. No.6,777,534 issued on Aug. 17, 2004, which is a continuation ofInternational Application No. PCT/US1998/026103 filed on Dec. 9, 1998,which designates the United States, and which claims benefit under 35U.S.C. §119(e) of U.S. Provisional Application No. 60/069,155 filed onDec. 9, 1997 and U.S. Provisional Application No. 60/069,687 filed onDec. 12, 1997, the entire contents of which are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work described herein was supported, in part, by National Instituteof Health grants CA37392 and CA45548. The U.S. Government has certainrights to the invention.

FIELD OF THE INVENTION

The present invention relates to vascular endothelial growth factor(VEGF). More particularly, the invention relates to antagonists of VEGFand use of those antagonists in the treatment of disorders that areassociated with VEGF.

BACKGROUND OF THE INVENTION

Blood vessels are the means by which oxygen and nutrients are suppliedto living tissues and waste products are removed from living tissue.Angiogenesis refers to the process by which new blood vessels areformed. See, for example, the review by Folkrnan and Shing, J. Biol.Chem. 267, 1093 1-1 0934 (1 992), Dvorak, et al., J. Exp. Med., 174,1275-1 278 (1 991). Thus, where appropriate, angiogenesis is a criticalbiological process. It is essential in reproduction, development andwound repair. However, inappropriate angiogenesis can have severenegative consequences. For example, it is only after many solid tumorsare vascularized as a result of angiogenesis that the tumors have asufficient supply of oxygen and nutrients that permit it to grow rapidlyand metastasize. Because maintaining the rate of angiogenesis in itsproper equilibrium is so critical to a range of functions, it must becarefully regulated in order to maintain health. The angiogenesisprocess is believed to begin with the degradation of the basementmembrane by proteases secreted from endothelial cells (EC) activated bymitogens such as vascular endothelial growth factor (VEGF) and basicfibroblast growth factor (bFGF). The cells migrate and proliferate,leading to the formation of solid endothelial cell sprouts into thestromal space, then, vascular loops are formed and capillary tubesdevelop with formation of tight junctions and deposition of new basementmembrane.

In adults, the proliferation rate of endothelial cells is typically lowcompared to other cell types in the body. The turnover time of thesecells can exceed one thousand days. Physiological exceptions in whichangiogenesis results in rapid proliferation typically occurs under tightregulation, such as found in the female reproduction system and duringwound healing.

The rate of angiogenesis involves a change in the local equilibriumbetween positive and negative regulators of the growth of microvessels.The therapeutic implications of angiogenic growth factors were firstdescribed by Folkman and colleagues over two decades ago (Folkman, N.Engl. J. Med., 285: 1 182-1 1 86 (1971)). Abnormal angiogenesis occurswhen the body loses at least some control of angiogenesis, resulting ineither excessive or insufficient blood vessel growth. For instance,conditions such as ulcers, strokes, and heart attacks may result fromthe absence of angiogenesis normally required for natural healing. Incontrast, excessive blood vessel proliferation can result in tumorgrowth, tumor spread, blindness, psoriasis and rheumatoid arthritis.

Thus, there are instances where a greater degree of angiogenesis isdesirable—increasing blood circulation, wound healing, and ulcerhealing. For example, recent investigations have established thefeasibility of using recombinant angiogenic growth factors, such asfibroblast growth factor (FGF) family (Yanagisawa-Miwa, et al., Science,257: 1401-1403 (1992) and Baffour, et al., J Vasc Surg, 16: 181-91(1992)), endothelial cell growth factor (ECGF) (Pu, et al., J. Surg Res,54575-83 (1993)), and more recently, vascular endothelial growth factor(VEGF) to expedite and/or augment collateral artery development inanimal models of myocardial and hindlimb ischemia (Takeshita, et al.,Circulation, 90:228-234 (1994) and Takeshita, et al., J. Clin Invest,93:662-70 (1994)).

Conversely, there are instances, where inhibition of angiogenesis isdesirable. For example, many diseases are driven by persistentunregulated angiogenesis, also sometimes referred to as“neovascularization.” In arthritis, new capillary blood vessels invadethe joint and destroy cartilage. In diabetes, new capillaries invade thevitreous, bleed, and cause blindness. Ocular neovascularization is themost common cause of blindness. Tumor growth and metastasis areangiogenesis-dependent. A tumor must continuously stimulate the growthof new capillary blood vessels for the tumor itself to grow.

There is mounting evidence that VEGF may be a major regulator ofangiogenesis (reviewed in Ferrara, et al., Endocr. Rev., 13, 18-32 (1992); Klagsbrun, et al., Curr. Biol., 3, 699-702 (1993); Klagsbrun, etal., Ferrara, et al., Biochem. Biophys. Res. Commun., 161, 85 1-858 (1989)). VEGF was initially purified from the conditioned media offolliculostellate cells (Ferrara, et al., Biochem. Biopsy. Res. Commun.,161,85 1-858 (1989)) and from a variety of tumor cell lines (Myoken, etal., Proc. Natl. Acad. Sci. USA, 88:5819-5823 (1991); Plouet, et al.,EMBO. J., 8:3801-3806 (1991)). VEGF was found to be identical tovascular permeability factor, a regulator of blood vessel permeabilitythat was purified from the conditioned medium of U937 cells at the sametime (Keck, et al., Science, 246: 1309-1 3 12 (1989)). VEGF is aspecific mitogen for endothelial cells (EC) in vitro and a potentangiogenic factor in vivo. The expression of VEGF is up-regulated intissue undergoing vascularization during embryogenesis and the femalereproductive cycle (Brier, et al., Development, 114:521-532 (1992);Shweiki, et al., J. Clin. Invest., 91:2235-2243 (1993)). High levels ofVEGF are expressed in various types of tumors, but not in normal tissue,in response to tumor-induced hypoxia (Shweiki, et al., Nature359:843-846 (1992); Dvorak et al., J. Exp. Med., 174:1275-1278 (1991);Plate, et al., Cancer Res., 535822-5827; Lkea, et al., J. Biol. Chem.,270:19761-19766 (1986)). Treatment of tumors with monoclonal antibodiesdirected against VEGF resulted in a dramatic reduction in tumor mass dueto the suppression of tumor angiogenesis (Kim, et al., Nature,382:841-844 (1993)). VEGF appears to play a principle role in manypathological states and processes related to neovascularization.Regulation of VEGF expression in affected tissues could therefore be keyin treatment or prevention of VEGF inducedneovascularization/angiogenesis.

VEGF is a secreted 40-45K homodimer (Tischer E, et. al., J. Biol. Chem.266: 11947-11954 (1991). It is a member of an expanding family thatincludes placenta derived growth factor (PlGF), VEGF-B, VEGF-C, VEGF-Dand VEGF-E (Olofsson et. al., Proc. Natl. Acad. Sci. USA 93:2576-258 1(1 996), Joukov et. al., EMBO J. 15:290-298 (1996), Achen et. al., Proc.Natl. Acad. Sci. USA 95548-553 (1998). Ogawa et. al., J Biol. Chem. 273:3 1273-31282 (1998)). VEGF exists in a number of different isoforms thatare produced by alternative splicing from a single gene containing eightexons (Ferrara, et al., Endocr. Rev., 13: 18-32 (1 992); Tischer, etal., J Biol. Chem., 806: 1 1947-1 1954 (1991); Ferrara, et al., TrendsCardio Med., 3:244-250 (1993); Polterak, et al., J. Biol. Chem., 272:7151-7 158 (1 997)). Human VEGF isoforms consists of monomers of 121, 145,165, 189, and 206 amino acids, each capable of making an activehomodimer (Polterak et al., J. Biol. Chem, 272:715 1-7158 (1997); Houck,et al., Mol. Endocrinol., 8: 1806-1 8 14 (1991)). The VEGF₁₂₁ andVEGF₁₆₅ isoforms are the most abundant. VEGF₁₂₁ is the only VEGFisoforms that does not bind to heparin and is totally secreted into theculture medium. VEGF₁₆₅ is functionally different than VEGF₁₂₁, in thatit binds to heparin and cell surface heparin sulfate proteoglycans(HSPGs) and is only partially released into the culture medium (Houck,et al., J. Biol. Chem., 247:28031-28037 (1992); Park, et al., Mol. Biol.Chem., 4: 1317-1326 (1993)). The remaining isoforms are entirelyassociated with cell surface and extracellular matrix HSPGs (Houck, etal., J. Biol. Chem., 247:2803 1-28037(1992); Park, et al., Mol. Biol.Chem., 4: 13 17-1326 (1993)).

VEGF receptor tyrosine kinases, KDR/Flk-1 and/or Flt-1, are mostlyexpressed by EC (Terman, et al., Biochem. Biophys. Res. Commun., 187:1579-1 586 (1992); Shibuya, et al., Oncogene, 5519-524 (1990); De Vries,et al., Science, 265:989-991 (1992); Gitay-Goran, et al., J. Biol.Chem., 287:6003-6096 (1992); Jakeman, et al., J Clin. Invest.,89:244-253 (1992)). It appears that VEGF activities such asmitogenicity, chemotaxis, and induction of morphological changes aremediated by KDR/Flk-1 but not Flt-1, even though both receptors undergophosphorylation upon binding of VEGF (Millauer, et al., Cell, 72:835-846(1993); Waltenberger, et al., J. Biol. Chem., 269:26988-26995 (1994);Seetharam, et al., Oncogene, 10: 135-147 (1995); Yoshida, et al., GrowthFactors, 7: 131-138 (1996)). Recently, Soker et al., identified a newVEGF receptor which is expressed on EC and various tumor-derived celllines such as breast cancer-derived MDA-MB-23 1 (231) cells (Soker, etal., J. Biol. Chem., 271 5761-5767 (1996)). This receptor requires theVEGF isoform to contain the portion encoded by exon 7. For example,although both VEGF₁₂₁ and VEGF₁₆₅R bind to KDR/Flk-1 and Flt-1, onlyVEGF₁₆₅ binds to the new receptor. Thus, this is an isoform-specificreceptor and has been named the VEGF₁₆₅ receptor (VEGF₁₆₅R). It willalso bind the 189 and 206 isoforms. In structure-function analysis, itwas shown directly that VEGF₁₆₅ binds to VEGF₁₆₅R via its exon 7-encodeddomain which is absent in VEGF121 (Soker, et al., J. Biol. Chem., 2715761-5767 (1996)). However, the function of the receptor was unclear.

The current treatment of angiogenic diseases is inadequate. Agents whichprevent continued angiogenesis, e.g, drugs (TNP-470), monoclonalantibodies, antisense nucleic acids and proteins (angiostatin andendostatin) are currently being tested. See, Battegay, J. Mol. Med., 73,333-346 (1995); Hanahan et al., Cell, 86, 353-364 (1996); Folkman, N.Engl. J. Med., 333, 1757-1763 (1995). Although preliminary results withthe antiangiogenic proteins are promising, they are relatively large insize and thus difficult to use and produce. Moreover, proteins aresubject to enzymatic degradation. Thus, new agents that inhibitangiogenesis are needed. New antiangiogenic proteins or peptides thatshow improvement in size, ease of production, stability and/or potencywould be desirable.

SUMMARY OF THE INVENTION

We have discovered that a portion of the seventh exon of VEGFI65 acts asan antagonist to all VEGF isoforms, which is surprising since not allforms of VEGF have exon 7. For example, we have prepared a glutathioneS-transferase (GST) fusion protein containing a peptide corresponding tothe 44 amino acids encoded by exon 7 and the first cystein of thepeptide encoded by exon 8 (amino acids 116-160 of VEGF₁₆₅ (SEQ ID NO:I)). This fusion protein inhibited the binding of ¹²⁵I-VEGF₁₆₅ toreceptors on human umbilical cord vein-derived EC (HUVEC) and on 23 1cells. The inhibitory activity was localized to the C-terminal portionof the exon 7-encoded domain (amino acids 22-44). Furthermore, thefusion protein inhibited VEGF-induced proliferation of HUVEC. The fusionprotein also inhibits VEGF₁₂₁-induced mitogenicity, which was anunexpected result considering that VEGF₁₂₁ does not contain exon 7.Thus, the polypeptides of the present invention are antagonists againstthe major isoforms of VEGF and can be used to treat diseases andconditions associated with VEGF-induced neovascularization orangiogenesis.

In addition, while not wishing to be bound by theory, it is believedthat VEGF is directly associated with a number of cancers expressing theVEGF₁₆₅R/NP-1 (Soker, et al., Cell 92,735-745 (1998)), and thatinhibition of VEGF binding to this receptor can be used to treat suchcancers.

The present invention provides a polypeptide having a portion of SEQ IDNO: 1 having VEGF antagonist activity as determined, for example, by thehuman umbilical vein endothelial cell (HUVEC) proliferation assay usingVEGF165 as set forth below in the Examples. Preferably, the portion hasat least a 25% reduction in HUVEC proliferation, more preferably a 50%reduction, even more preferably a 75% reduction, most preferably a 95%reduction. Preferably, the portion has an even number of cysteineresidues.

VEGF antagonist activity may also be determined by inhibition of bindingof labeled VEGF165 to VEGF165R as disclosed in Soker et al., J. Biol.Chem. 271,5761-5767 (1996)) and forth below in the Examples. Preferably,the portion inhibits binding by at least 25%, more preferably 5096, mostpreferably 75%.

The present invention further provides polypeptides comprising SEQ IDNO: 2 (CSCKNTDSRCKARQLELNERTCRC) or a portion thereof having VEGFantagonist activity as determined, for example, by the human umbilicalvein endothelial cell (HUVEC) proliferation assay using VEGF165 as setforth below in the Examples. Preferably, the portion has at least a 25%reduction in HUVEC proliferation, more preferably a 50% reduction, evenmore preferably a 75% reduction, most preferably a 95% reduction.Preferably, the portion has an even number of cysteine residues.

One preferred polypeptide of the present invention has the structure ofthe following formula (I):

(SEQ. ID NO: 3) (X₁-(CSCKNTDSRCKARQLELNERT)-X₂) Iwherein X₁ is H, or any portion of amino acids 2-21 of SEQ ID NO: 1. Forexample, amino acid 3-21,4-21,5-21,6-21, etc. of SEQ. ID NO: 1. And X₂is H or C, CR, RC or CRC. The polypeptides of formula (I) have VEGFantagonist activity as determined, for example, by the human umbilicalvein endothelial cell (HUVEC) proliferation assay using VEGF₁₆₅ as setforth below in the Examples. Preferably, the polypeptide has at least a25% reduction in HUVEC proliferation, more preferably a 50% reduction,even more preferably a 75% reduction, most preferably a 95% reduction.Preferably, the polypeptide has an even number of cysteine residues. Thepolypeptides of formula (I) include analogs.

“Analogs” refers to a polypeptide differing from the sequence of one ofthe peptides of the invention but which still exhibits at least 50% ofthe VEGF antagonist activity of the polypeptide of SEQ ID NO: 2 in thehuman umbilical vein endothelial cell (HUVEC) proliferation assay usingVEGF₁₆₅ as set forth below in the Examples. Preferably, the analogexhibits 75% of the VEGF antagonist activity of the polypeptide of SEQID NO: 2, most preferably 95%. The differences are preferablyconservative amino acid substitutions, in which an amino acid isreplaced with another naturally occurring amino acid of similarcharacter. For example, the following substitutions are considered“conservative”: Gly⇄Ala; Val⇄Ile; Asp⇄Glu; Lys⇄Arg; Asn⇄Gln; andPhe⇄Trp⇄Tyr. Nonconservative changes are generally substitutions of oneof the above amino acids with an amino acid from a different group(e.g., substituting Asn for Glu), or substituting Cys, Met, His, or Profor any of the above amino acids.

In preferred forms, the polypeptides of the present invention are partof a fusion protein or conjugated to a moiety to enhance purification,increase stability and/or to provide a biological activity.

In another embodiment, the polypeptides of the present invention, eitheralone, or as part of a fusion protein, are used to target cellsexpressing the VEGF₁₆₅R/NP-1. This targeting can be used for diagnosticas well as therapeutic applications. For example, for diagnosticpurposes the polypeptide is radiolabeled and used to detect cellsexpressing the VEGF165R/NP-1. We have discovered that expression of thereceptor has a high correlation to disease state in a number of cancers,such as prostate and breast, particularly metastatic cancers.Accordingly, in a further embodiment, the polypeptide can be used in aprognostic manner for particular cancers.

For therapeutic applications, the polypeptide can be used to deliveragents to cells expressing the VEGF₁₆₅R/NP-1. For example, thepolypeptides can be used as carriers to deliver a desired chemical orcytotoxic moiety to the cells. The cytotoxic moiety may be a cytotoxicdrug or an enzymatically active toxin of bacterial, fungal or plantorigin, or an enzymatically active polypeptide chain or fragment (“Achain”) of such a toxin. Enzymatically active toxins and fragmentsthereof are preferred and are exemplified by diphtheria toxin Afragment, non-binding active fragments of diphtheria toxin, exotoxin A(from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin Achain, alphasarcin, certain Aleurites fordii proteins, certain Dianthinproteins, Phytolacca americana proteins (PAP, PAP11 and PAP-S),Momordica charantia inhibitor, curcin, crotin, Saponaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin.Ricin A chain, Pseudomonas aeruginosa exotoxin A and PAP are preferred.

The invention further provides a method of treating a disease ordisorder/condition associated with VEGF-induced neovascularization orangiogenesis. As used herein, the term “neovascularization” refers tothe growth of blood vessels and capillaries. Diseases, disorders, orconditions, associated with VEGF-induced neovascularization orangiogenesis, include, but are not limited to retinalneovascularization, hemagiomas, solid tumor growth, leukemia,metastasis, psoriasis, neovascular glaucoma, diabetic retinopathy,rheumatoid arthritis, osteoarthritis, endometriosis, musculardegeneration and retinopathy of prematurity (ROP).

In the methods of the present invention, a therapeutic amount of apolypeptide of the invention is administered to a host, e.g., human orother mammal, having a disease or condition, associated with VEGF orhaving a tumor expressing VEGF165R/NP-1. Methods for detecting theexpression of VEGF165R/NP-1 are set forth in Soker, et al., Cell92:735-745 (1998).

The invention also provides a composition comprising an effective amountof a polypeptide of the invention in combination with a pharmaceuticallyacceptable carrier.

Other aspects of the invention are disclosed infia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Cross-linking of 1 ¹²⁵I-VEGF₁₆₅. ¹²⁵I-VEGF₁₂₁ and ¹²⁵I-GST-EX 7to HUVEC. ¹²⁵I-VEGF₁₆₅ (5 ng/ml) (lane 1) or ¹²⁵I-VEGF₁₂₁ (10 ng/ml)(lane 2) or ¹²⁵I-GST-EX 7 (50 nglml) (lane 3) were bound to subconfluentcultures of HUVEC in 6-cm dishes. The binding was carried out in thepresence of 1 ug/ml heparin. At the end of a 2-H incubation, each¹²⁵I-VEGF isoform was chemically cross-linked to the cell surface. Thecells were lysed and proteins were resolved by 6% SDS-PAGE. Thepolyacrylamide gel was dried and exposed to x-ray film.

FIG. 2. HUVEC proliferation in response to VEGF₁₆₅ and VEGF₁₂₁ HUVECwere cultured in 96-well dishes (5,000 cell/well) for 24 h. Increasingamounts of VEGF₁₆₅ (closed circles) or VEGF₁₂₁ (open circles) were addedto the medium, and the cells were incubated for 3 more days. DNAsynthesis based on the incorporation of [3H] thymidine into HUVEC DNAwas measured as described in the Examples. The results represent theaverage counts in three wells, and the standard deviations weredetermined.

FIGS. 3A-3C. Inhibition of ¹²⁵I-VEGF₁₆₅ binding to HUVEC, MDA MB 231cells, and PAE-KDR cells by GST-EX 7+8. ¹²⁵I-VEGF₁₆₅ (5 ng,/ml) wasbound to subconfluent cultures of HUVEC (3A), MDA MB 23 1 cells (3B),and PAE-KDR cells (3C) in 48-well dishes in the presence of increasingamounts of GST-Ex 7+8 (closed 20 square) or control GST protein (opensquares). At the end of a 2-h incubation, the cells were washed andlysed, and the cell-associated radioactivity was determined with aγ-counter. The counts obtained are expressed as the percentage of thecounts obtained in the presence of PBS without addition of GST or fusionprotein.

FIG. 4. GST-EX 7+8 fusion protein inhibits cross-linking of ¹²⁵I-VEGF₁₆₅to ¹²⁵I-VEGF₁₆₅R and to KDR/Flk-1. ¹²⁵I-VEGF₁₆₅ (5 ng/ml) was bound tosubconfluent cultures of HUVEC (lanes 1 and 2) and MDA-MB-23 1 cells(lanes 3 and 4) in 6-cm dishes. The binding was carried out in thepresence (lanes 2 and 4) or the absence (lanes 1 and 3) of 15 ug/mlGST-Ex 7+8. Heparin (1 ug/ml) was added to each dish. At the end of a2-h incubation, ¹²⁵I-VEGF₁₆₅ was chemically cross-linked to the cellsurface. The cells were lysed, and proteins were resolved by 6%SDS-PAGE. The gel was dried and exposed to x-ray film.

FIGS. 5A and 5B. Localization of a core inhibitory region within exon 7.GST-Ex 7 fusion proteins containing full-length exon 7-encoded domain ortruncations at the N-terminal and C-terminal ends were prepared asdescribed in the Examples. ¹²⁵I-VEGF₁₆₅ (5 ng/ml) was bound tosubconfluent HUVEC cultures as described in FIG. 3, in the presence ofincreasing concentrations of the GST fusion proteins. At the end of a2-h incubation, the cells were washed and lysed, and the cell-associatedradioactivity was determined with a y counter. The counts obtained areexpressed as percentage of the counts obtained in the presence of PBSwithout fusion protein B, the amino acid sequences of VEGF exon 7derivatives. These derivatives were prepared to contain the firstcysteine residue of exon 8 at their C termini to keep an even number ofcysteine residue.

FIG. 6. GST-Ex 7+8 fusion protein inhibits VEGF₁₆₅-stimulated HUVECproliferation. HUVEC were cultured in 96-well dishes (5,000 cell/well)as in FIG. 2. Increasing concentrations of VEGF165 (open circles),together with 15 ug/ml GST-Ex 7+8 (closed circles) or 25 ug/ml GST(squares), were added to the medium, and the cells were incubated for 4more days. DNA synthesis was measured in HUVEC as described in FIG. 2.The results represent the average counts of three wells, and thestandard deviations were determined.

FIG. 7. GST-Ex 7+8 fusion protein inhibits VEGF₁₆₅ andVEGF₁₂₁-stimulated HUVEC proliferation. Increasing concentration ofVEGF₁₆₅ (circles) or VEGF₁₂₁ (square) with 15 ug/ml GST-Ex 7+8 (closedsymbols) or without GST-Ex 7+8 (open symbols) were added to HUVEC, and[*H]thymidine incorporation into the DNA was measured as in FIG. 2. Theresults represent the average counts of three wells and the standarddeviations were determined.

FIG. 8. GST-Ex 7+8 fusion protein inhibits cross-linking of ¹²⁵I-VEGF₁₂₁to KDR/Flk-1 of HUVEC ¹²⁵I-VEGF₁₂₁ (2˜0 ng/ml) was bound to subconfluentcultures of HUVEC in 6-cm dishes. The binding was carried out in thepresence (lane 2) or the absence (lane 1) of 15 ug/ml GST-Ex 7+8.Heparin (1 ug/ml) was added to each dish. At the end of a 2-hincubation, ¹²⁵I-VEGF was chemically cross-linked to the cell surface.The cells were lysed, and proteins were resolved by 6% SDS-PAGE. The gelwas dried and exposed to x-ray film.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated polypeptides having VEGFantagonist activity, nucleic acids encoding peptides, pharmaceuticalcompositions comprising the polypeptides and nucleic acids and methodsfor treating diseases or disorders associated with VEGF, e.g., tumorsthat express VEGF₁₆₅ R/NP-1 and VEGF induced angiogenesis. Thepolypeptides of the invention include polypeptides comprising a portionof SEQ ID NO: 1 having VEGF antagonist activity, polypeptides comprisingSEQ ID NO: 2 (CSCKNTDSRCKARQLELNERTCRC) or a portion thereof having VEGFantagonist activity, and a polypeptide having the structure of formula(I), set forth above. The present invention further includes analogs andderivatives of these polypeptides having VEGF antagonist activity. TheDNA sequence encoding exon 7 and exon 8 are set forth in the sequencelisting as SEQ ID NOS: 17 and 18 respectively.

VEGF antagonist activity can be determined using techniques known in theart. For example, VEGF antagonist activity can be determined by lookingat a wild type VEGF activity and comparing the inhibition or reductionof such activity when the antagonist polypeptide is used. Thepolypeptide of SEQ ID NO: 2 can be used as a standard. One can use anyVEGF activity. For example, one can use the human umbilical veinendothelial cell (HUVEC) proliferation assay using VEGFI65 as set forthbelow in the Examples. Preferably, the portion has at least a 25%reduction in HUVEC proliferation, more preferably a 50% reduction, evenmore preferably a 75% reduction, most preferably a 95% reduction.Preferably, the portion has an even number of cysteine residues.

VEGF antagonist activity may also be determined by inhibition of bindingof labeled VEGFI65 to VEGF165R as disclosed in Soker et al., J. Biol.Chem. 271, 5761-5767 (1996)) and forth below in the Examples.Preferably, the portion inhibits binding by at least 25%, morepreferably 50%, most preferably 75%.

The ability of the VEGF antagonist polypeptides to influenceangiogenesis can also be determined using a number of know in vivo andin vitro assays. Such assays are disclosed in Jain et al., NatureMedicine 3, 1203-1 208(1997), the disclosure of which is hereinincorporated by reference. For example, assays for the ability toinhibit angiogenesis in vivo include the chick chorioallantoic membraneassay and mouse, rat or rabbit corneal pocket assays. See, Polverini etal., 1991, Methods Enzymol. 198: 440-450. According the corneal pocketassays, a tumor of choice is implanted into the cornea of the testanimal in the form of a corneal pocket. The potential angiogenesisinhibitor is applied to the corneal pocket and the corneal pocket isroutinely examined for neovascularization.

As used herein, a “derivative” of a VEGF antagonist polypeptide is apolypeptide in which one or more physical, chemical, or biologicalproperties has been altered. Such modifications include, but are notlimited to: amino acid substitutions, modifications, additions ordeletions; alterations in the pattern of lipidation, glycosylation orphosphorylation; reactions of free amino, carboxyl, or hydroxyl sidegroups of the amino acid residues present in the polypeptide with otherorganic and non-organic molecules; and other modifications, any of whichmay result in changes in primary, secondary or tertiary structure. Yetsuch a derivative will exhibit at least one of the aforementioned VEGFantagonist activities.

The polypeptides of the invention are preferably produced by recombinantmethods. See the procedures disclosed in Example 1, which follows. Awide variety of molecular and biochemical methods are available forgenerating and expressing the polypeptides of the present invention; seee.g. the procedures disclosed in Molecular Cloning, A Laboratory Manual(2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), CurrentProtocols in Molecular Biology (Eds. Aufbbel, Brent, Kingston, More,Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, NY,N.Y. 1992) or other procedures that are otherwise known in the art. Forexample, the polypeptides of the invention may be obtained by chemicalsynthesis, expression in bacteria such as E. coli and eukaryotes such asyeast, baculovirus, or mammalian cell-based expression systems, etc.,depending on the size, nature and quantity of the polypeptide.

The term “isolated” means that the polypeptide is removed from itsoriginal environment (e.g., the native VEGF molecule). For example, anaturally-occurring polynucleotides or polypeptides present in a livinganimal is not isolated, but the same polynucleotides or DNA orpolypeptides, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

Where it is desired to express a polypeptide of the invention anysuitable system can be used. The general nature of suitable vectors,expression vectors and constructions therefor will be apparent to thoseskilled in the art.

Suitable expression vectors may be based on phages or plasmids, both ofwhich are generally host-specific, although these can often beengineered for other hosts. Other suitable vectors include cosmids andretroviruses, and any other vehicles, which may or may not be specificfor a given system. Control sequences, such as recognition, promoter,operator, inducer, terminator and other sequences essential and/oruseful in the regulation of expression, will be readily apparent tothose skilled in the art.

Correct preparation of nucleotide sequences may be confirmed, forexample, by the method of Sanger et al. (Proc. Natl. Acad. Sci. USA745463-7(1977)).

A DNA fragment encoding the polypeptide of the invention, the receptoror fragment thereof, may readily be inserted into a suitable vector.Ideally, the receiving vector has suitable restriction sites for ease ofinsertion, but blunt-end ligation, for example, may also be used,although this may lead to uncertainty over reading frame and directionof insertion. In such an instance, it is a matter of course to testtransformants for expression, 1 in 6 of which should have the correctreading frame. Suitable vectors may be selected as a matter of course bythose skilled in the art according to the expression system desired.

By transforming a suitable organism or, preferably, eukaryotic cellline, such as HeLa, with the plasmid obtained, selecting thetransformant with ampicillin or by other suitable means if required, andadding tryptophan or other suitable promoter-inducer (such asindoleacrylic acid) if necessary, the desired polypeptide or protein maybe expressed. The extent of expression may be analyzed by SDSpolyacrylamide gel electrophoresis-SDS-PAGE (Lemelli, Nature 227:680-685(1970)).

Suitable methods for growing and transforming cultures etc. are usefullyillustrated in, for example, Maniatis (Molecular Cloning, A LaboratoryNotebook, Maniatis et al. (eds.), Cold Spring Harbor Labs, N.Y. (1989)).

Cultures useful for production of polypeptides or proteins may suitablybe cultures of any living cells, and may vary from prokaryoticexpression systems up to eukaryotic expression systems. One preferredprokaryotic system is that of E. coli, owing to its ease ofmanipulation. However, it is also possible to use a higher system, suchas a mammalian cell line, for expression of an eukaryotic protein.Currently preferred cell lines for transient expression are the HeLa andCos cell lines. Other expression systems include the Chinese HamsterOvary (CHO) cell line and the baculovirus system.

Other expression systems which may be employed include streptomycetes,for example, and yeasts, such as Saccharomyces spp., especially S.cerevisiae. Any system may be used as desired, generally depending onwhat is required by the operator. Suitable systems may also be used toamplify the genetic material, but it is generally convenient to use E.coli for this purpose when only proliferation of the DNA is required.

The polypeptides and proteins may be isolated from the fermentation orcell culture and purified using any of a variety of conventional methodsincluding: liquid chromatography such as normal or reversed phase, usingHPLC, FPLC and the like; affinity chromatography (such as with inorganicligands or monoclonal antibodies); size exclusion chromatography;immobilized metal chelate chromatography; gel electrophoresis; and thelike. One of skill in the art may select the most appropriate isolationand purification techniques without departing from the scope of thisinvention.

The polypeptides may also be generated by any of several chemicaltechniques. For example, they may be prepared using the solid-phasesynthetic technique originally described by R. B. Merrifield, “SolidPhase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide”, J. Am.Chem. Soc., 83, pp. 2149-54 (1963), or they may be prepared by synthesisin solution. A summary of peptide synthesis techniques may be found inE. Gross & H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis,5Biology; Modem Techniques Of Peptide And Amino Acid Analysis, JohnWiley & Sons, (1981) and M. Bodanszky, Principles Of Peptide Synthesis,Springer-Verlag (1984).

As discussed above, one method of treatment involves attachment of asuitable toxin to the peptides which then target the area of the tumor.Such toxins are well known in the art, and may comprise toxicradioisotopes, heavy metals, enzymes and complement activators, as wellas such natural toxins as ricin which are capable of acting at the levelof only one or two molecules per cell. It may also be possible to usesuch a technique to deliver localized doses of suitable physiologicallyactive compounds, which may be used, for example, to treat cancers.

Where the present invention provides for the administration of, forexample, peptides to patient, then this may be by any suitable route. Ifthe tumor is still thought to be, or diagnosed as, localized, then anappropriate method of administration may be by injection direct to thesite. Administration may also be by injection, including subcutaneous,intramuscular, intravenous and intradermal injections.

Formulations may be any that are appropriate to the route ofadministration, and will be apparent to those skilled in the art. Theformulations may contain a suitable carrier, such as saline, and mayalso comprise bulking agents, other medicinal preparations, adjuvantsand any other suitable pharmaceutical ingredients. Catheters are anotherpreferred mode of administration.

The term “effective amount” refers to an amount of VEGF antagonistpolypeptide or nucleic acid encoding the polypeptide sufficient toexhibit a detectable therapeutic effect. The therapeutic effect mayinclude, for example, without limitation, inhibiting the growth ofundesired tissue or malignant cells, inhibiting inappropriateangiogenesis (neovascularization), limiting tissue damage caused bychronic inflammation, inhibition of tumor cell growth, and the like. Theprecise effective amount for a subject will depend upon the subject'ssize and health, the nature and severity of the condition to be treated,and the like. Thus, it is not possible to specify an exact effectiveamount in advance. However, the effective amount for a given situationcan be determined by routine experimentation based on the informationprovided herein.

The term “pharmaceutically acceptable” refers to compounds andcompositions which may be administered to mammals without unduetoxicity. Exemplary pharmaceutically acceptable salts include mineralacid salts such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like.

The VEGF antagonist polypeptides are administered orally, topically, orby parenteral means, including subcutaneous and intramuscular injection,implantation of sustained release depots, intravenous injection,intranasal administration, and the like. Accordingly, VEGF antagonistsmay be administered as a pharmaceutical composition comprising a VEGFantagonist in combination with a pharmaceutically acceptable carrier.Such compositions may be aqueous solutions, emulsions, creams,ointments, suspensions, gels, liposomal suspensions, and the like.Suitable carriers (excipients) include water, saline, Ringer's solution,dextrose solution, and solutions of ethanol, glucose, sucrose, dextran,mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate,acetate, gelatin, collagen, Carbopol Registered™, vegetable oils, andthe like. One may additionally include suitable preservatives,stabilizers, antioxidants, antimicrobials, and buffering agents, forexample, BHA, BHT, citric acid, ascorbic acid, tetracycline, and thelike. Cream or ointment bases useful in formulation include lanolin,Silvadene Registered™ (Marion), Aquaphor Registered™ (DukeLaboratories), and the like. Other topical formulations includeaerosols, bandages, and other wound dressings. Alternatively one mayincorporate or encapsulate the VEGF antagonist in a suitable polymermatrix or membrane, thus providing a sustained release delivery devicesuitable for implantation near the site to be treated locally. Otherdevices include indwelling catheters and devices such as the AlzetRegistered™ minipump. Ophthalmic preparations may be formulated usingcommercially available vehicles such as Sorbi-care Registered™(Allergan), Neodecadron Registered™ (Merck, Sharp & Dohrne), LacrilubeRegistered™, and the like, or may employ topical preparations such asthat described in U.S. Pat. No. 5,124,155, incorporated herein byreference. Further, one may provide a VEGF antagonist in solid form,especially as a lyophilized powder. Lyophilized formulations typicallycontain stabilizing and bulking agents, for example human serum albumin,sucrose, mannitol, and the like. A thorough discussion ofpharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co.).

The antagonist polypeptides of the present invention can be usedtopically or intravascularly. For topical applications the formulationwould be applied directly at a rate of about 10 ng to about 1mg/cm2/day. For intravenous applications, the inhibitor is used at arate of about 1 mg to about 10 mg/kg/day of body weight. For internaluse, the formulation may be released directly into the region to betreated either from implanted slow release polymeric material or fromslow release pumps or repeated injections. The release rate in eithercase is about 100 ng to about 100 mg/day/cm3.

The VEGF antagonist polypeptides of the invention can be combined with atherapeutically effective amount of another molecule which negativelyregulates angiogenesis which may be, but is not limited to, TNP-470,platelet factor 4, thrombospondin-1, tissue inhibitors ofmetalloproteases (TIMPI and TIMP2), prolactin (1 6-Kd fragment),angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF solublereceptor, transforming growth factor P, interferon alfa, soluble KDR andFLT-201 receptors and placental proliferin-related protein.

A VEGF antagonist polypeptide of the invention may also be combined withchemotherapeutic agents.

Diseases, disorders, or conditions, associated with abnormalangiogenesis or neovascularization, and can be treated with atherapeutic compound of the invention include, but are not limited toretinal neovascularization, tumor growth, hemagioma, solid tumors,leukemia, metastasis, psoriasis, neovascular glaucoma, diabeticretinopathy, arthritis, endometriosis, and retinopathy of prematurity(ROP).

Nucleic acid (e.g., DNA) encoding a VEGF antagonist polypeptide of theinvention can be delivered to a host by any method known to those ofskill in the art to treat disorders associated with VEGF. A preferredembodiment of the present invention relates to methods of inhibitingangiogenesis of solid tumors to prevent further tumor growth andeventual metastasis. To this end, any solid tumor or the regionsurrounding the tumor accessible to gene transfer will be a target forthe disclosed therapeutic applications. A DNA encoding a VEGF antagonistpolypeptide of the invention or a derivative or analog thereof, housedwithin a recombinant viral- or non-viral-based gene transfer system maybe directed to target cells within proximity of the tumor by any numberof procedures known in the art, including but not limited to (a)surgical procedures coupled with administration of an effective amountof the DNA to the site in and around the tumor (involving initialremoval of a portion or the entire tumor, if possible); (b) injection ofthe gene transfer vehicle directly into or adjacent to the site of thetumor; and, (c) localized or systemic delivery of the gene transfervector and/or gene product using techniques known in the art.

Therefore, any solid tumor which contains VEGF or VEGF165R/NP1- or NP-2expressing cells will be a potential target for treatment. Examples, butby no means listed as a limitation, of solid tumors which will beparticularly vulnerable to gene therapy applications are (a) neoplasmsof the central nervous system such as, but again not necessarily limitedto glioblastomas, astrocytomas, neuroblastomas, meningiomas,ependymomas; (b) cancers of hormone-dependent, tissues such as prostate,testicles, uterus, cervix, ovary, mammary carcinomas including but notlimited to carcinoma in situ, medullary carcinoma, tubular carcinoma,invasive (infiltrating) carcinomas and mucinous carcinomas; (c)melanomas, including but not limited to cutaneous and ocular melanomas;(d) cancers of the lung which at least include squamous cell carcinoma,spindle carcinoma, small cell carcinoma, adenocarcinoma and large cellcarcinoma; and (e) cancers of the gastrointestinal system such asesophageal, stomach, small intestine, colon, colorectal, rectal and analregion which at least include adenocarcinomas of the large bowel.

Expression vectors are defined herein as DNA sequences that are requiredfor the transcription of cloned copies of genes and the translation oftheir rnRNAs in an appropriate host. Such vectors can be used to expresseukaryotic genes in a variety of hosts such as bacteria, bluegreenalgae, fungal cells, yeast cells, plant cells, insect cells and animalcells.

Specifically designed vectors allow the shuttling of DNA between hostssuch as bacteria-yeast or bacteria-animal or bacteria-insect cells. Anappropriately constructed expression vector should contain: an origin ofreplication for autonomous replication in host cells, selectablemarkers, a limited number of useful restriction enzyme sites, apotential for high copy number, and active promoters. A promoter isdefined as a DNA sequence that directs RNA polymerase to bind to DNA andinitiate RNA synthesis. A strong promoter is one which causes rnRNAs tobe initiated at high frequency.

Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to expressrecombinant VEGF antagonists in mammalian cells. Commercially availablemammalian expression vectors which may be suitable for recombinantexpression, include but are not limited to, pcDNA3.1 (Invitrogen),pBlueBacHis2 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39(New England BioLabs), pcDNAI, pcDNAlarnp (Invitrogen), pcDNA3(Invitrogen), pMC lneo (Stratagene), pXT1 (Stratagene); pSG5(Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 371 1 O),pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 3 7 199), pRSVneo(ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and ?LZD35(ATCC 37565).

DNA encoding a VEGF antagonist of the invention may also be cloned intoan expression vector for expression in a recombinant host cell.Recombinant host cells may be prokaryotic or eukaryotic, including butnot limited to bacteria, yeast, mammalian cells including but notlimited to cell lines of human, bovine, porcine, monkey and rodentorigin, and insect cells including but not limited to drosophila, moth,mosquito and armyworm derived cell lines. The expression vector may beintroduced into host cells via any one of a number of techniquesincluding but not limited to transformation, transfection, AdlpolylysineDNA complexes, protoplast fusion, and electroporation. Cell linesderived from mammalian species which may be suitable and which arecommercially available, include but are not limited to, CV-1 (ATCC CCL70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61),3T3 (ATCC CCL 92), NIW3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C 1271(ATCC CRL 161 6), BSC-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171) and HEK293 cells. Insect cell lines which may be suitable and are commerciallyavailable include but are not limited to 3M-S (ATCC CRL 8851) moth (ATCCCCL 80) mosquito (ATCC CCL 194 and 195; ATCC CRL 1660 and 1591) andarmyworm (SB, ATCC CRL 171 1).

A DNA fragment encoding a VEGF antagonist polypeptide may be deliveredeither systemically or to target cells in the proximity of a solid tumorof the mammalian host by viral or non-viral based methods. Viral vectorsystems which may be utilized in the present invention include, but arenot limited to, (a) adenovirus vectors; (b) retrovirus vectors; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picarnovirus vectors; and (i) vaccinia virus vectors.

The recombinant virus or vector containing the DNA encoding the VEGFantagonist of the present invention is preferably administered to thehost by direct injection into a solid tumor and/or quiescent tissueproximal to the solid tumor, such as adipose or muscle tissue. It willof course be useful to transfect tumor cells in the region of targetedadipose and muscle tissue. Transient expression of the VEGF antagonistin these surrounding cells will result in a local extracellular increasein these peptides and will promote binding with VEGF receptors, thusinhibiting binding of VEGF to the receptors.

Non-viral vectors which are also suitable include DNA-lipid complexes,for example liposome-mediated or ligandl poly-L-Lysine conjugates, suchas asialoglycoprotein-mediated delivery systems (see, e.g., Felgner etal., 1994, J. Biol. Chem. 269: 2550-2561; Derossi et al., 1995, Restor.Neurol. Neuros. 8: 7-10; and Abcallah et al.,1995, Biol. Cell 85: 1-7).

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutica compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

The references cited throughout this application are herein incorporatedby reference.

The present invention is further illustrated by the following Examples.These Examples are provided to aid in the understanding of the inventionand are not construed as a limitation thereof.

EXAMPLE 1 Experimental Procedures Materials.

Human recombinant VEGF165 and VEGFIZl produced in Sf-21 insect cellsinfected with recombinant baculovirus encoding human VEGF165 or VEGFlZ1as described previously (Soker, et al., J. Biol. Chem., 271, 5761-5767(1996), Cohen, et al., Growth Factors, 7, 13 1-1 38 (1992)). VEGFI6$ waspurified from the conditioned medium of infected Sf-21 cells by heparinaffinity chromatography, and VEGFlzl was purified by anion exchangechromatography. Basic FGF was kindly provided by Dr. Judith Abraham(Scios, Sunnyvale, Calif.). Cell culture media were purchased from LifeTechnologies, Inc. 125I-Sodium was purchased from NEN Life ScienceProducts. Disuccinimidyl suberate and IODO-BEADS were purchased fromPierce. Glutathione-agarose, NAP-5 columns, and pGEX-2TK plasmid werepurchased from Pharmacia Biotech Inc. TSK-heparin columns were purchasedfrom TosoHaas (Tokyo, Japan). Molecular weight marker was purchased fromAmersham Corp. (IL). Porcine intestinal mucosal-derived heparin waspurchased from Sigma.

Cell Culture.

Human umbilical vein endothelial cells (HUVEC) were obtained from theAmerican Type Culture Collection (ATCC) (Rockville, Md.) and grown ongelatin-coated dishes in M-199 medium containing 20% fetal calf serum(FCS) and a mixture of glutamine, penicillin, and streptomycin (GPS).Basic FGF (1 nglml) was added to the culture medium every other day.Porcine endothelial cells (PAE), parental and transfected to expressKDRElk-1 (PAE-KDR), were kindly provided by Dr. Lema Claesson-Welsh andgrown in F12 medium containing 10% FCS and GPS as described(Waltenberger, et al., J. Biol. Chem., 269,26988-26995 (1994)).MDAMB-231 (231) cells were obtained from ATCC and grown in Bulbecoo'smodified Eagle's medium containing 10% FCS and GPS.

Endothelial Cell Proliferation Assay.

HUVEC were seeded in gelatin-coated 96-well dishes at 4,000 cells/200Ul/well in M-199 containing 5% FCS and GPS. After 24 H, VEGF isoformsand VEGF exon 7-GST fusion proteins were added to the wells at the sametime. The cells were incubated for 72 h, and [3H]thymidine (1 pC/ml) wasadded for 10-12 h. The medium was aspirated, and the cells weretrypsinized and harvested by an automatic cell harvester (TOMTEC) andloaded onto Filtermats (Wallac). The Filtermats were scanned and cpmwere determined by a MicroBeta counter (Wallac). The results representthe average of samples assayed in triplicate, and the standardderivations were determined. All experiments were repeated at leastthree times and similar results were obtained.

Radioiodination of VEGF.

The radioiodination of VEGF1 65 and VEGF 12 1 was carried out usingIODOBEADS according to the manufacturer's instructions. Briefly, oneIODO-BEAD was rinsed with 100 ul of 0.1 M sodium phosphate, pH 7.2,dried, and incubated with 125I-sodium (0.2 mCi/ug protein) in 100 ul of0.1 M sodium phosphate, pH 7.2, for 5 min at room temperature. VEGF (1-3ug) was added to the reaction mixture, and after 5 min the reaction wasstopped by removing the bead. The solution containing 125I-VEGF wasadjusted to 2 mglml gelatin and purified by size exclusionchromatography using a NAP-5 column that was pre-equilibrated with PBScontaining 2 mg/ml gelatin. Aliquots of the iodinated proteins werefrozen on dry ice and stored at −80° C. The specific activity rangedfrom 40,000 to 100,000 cpm/ng protein.

Binding and Cross-Linking of 125I-VEGF

Binding and cross-linking experiments using 125I-VEGF165 and125I-VEGF121 were performed at described previously (Gitay-Goren, etal., J. Biol. Chem., 287, 6003-6096 (1992), Soker, et al., J: Biol.Chem., 271,5761-5767 (1996)). VEGF binding was quantified by measuringthe cell-associated radioactivity in a y-counter (Beckman, Gamma 5500).The counts represent the average of three walls. All experiments wererepeated at least three times, and similar results were obtained.125I-VEGF cross-linked complexes were resolved by 6% SDS-PAGE, and thegels were exposed to a phosphor screen and scanned after 24 h by aPhosphorImager (Molecular Dynamics). Subsequently, the gels were exposedto x-ray film.

Preparation of GST-VEGF Exon 7 and 8 Fusion Proteins.

Different segments of exons 7 and 8 of VEGF were amplified by thepolymerase chain reaction from human VEGF cDNA using the followingprimers: exon 7+8 (Ex 7+8), CGGGATCCCCCCTGTGGGCCTTGCTC (SEQ ID NO:4) andGGAATTCTTACCGCTCGGCTTGTC (SEQ ID NO:5); exon 7 (Ex 7),CGGGATCCCCCTGTGGGCCTTGCTC (SEQ ID NO:6) and GGAATTCTTAACATCTGCAAGTACGTT(SEQ ID NO:7) and exon 7 with residues 1-10 deleted (Ex 7d-(1-1 O)),CGGGATCCCATTTGTTTGTACAAGAT (SEQ ID NO:8) and GGAATTCTTAACATCTGCAAGTACGTT(SEQ ID NO:9) exon 7 with residues 1-2 1 deleted (Ex 7d-(I-2 I)),CGGGATCCTGTTCCTGCAAAAACACAG (SEQ ID NO: 10) andGGAATTCTTAACATCTGCAAGTACGTT (SEQ ID NO: 1 1) exon 7 with residues 1-22deleted (Ex 7d-(1-22) deleted (Ex 7d-(1-22)), CGGGATCCTGCAAAAACACAG (SEQID NO: 12) and GGAATTCTTAACATCTGCAAGTACGTT (SEQ ID NO: 13), andGGAATTCTTAACATCTGCAAGTACGTT (SEQ ID NO: 14), and exon 7 with residues30-44 deleted (Ex 7d-(30-44)), CGGGATCCCCCTGTGGGCCTTGCTC (SEQ ID NO: 15)and GGAATTCTAGTCTGTGTTTTTGCA (SEQ ID NO: 16).

The amplified products were digested with BamHI and EcoRI restrictionenzymes and cloned into the vector pGEK-2TK (Pharmacia Biotech Inc.)encoding GST (Smith, et al., Gene (Amst.), 87,3 1-40 (1988)) to yieldthe plasmid p2TK-exon 7+8 and its derivatives. Escherichia coli (DH4a)were transformed with p2TK-exon 7+8 and derivatives to produce GSTfusion proteins (see FIG. 5B for sequences). Bacterial lysates weresubsequently separated by a glutathione-agarose affinity chromatography(Smith, et al., Gene (Amst.), 87,3 1-40 (1 988)). Samples eluted fromglutatione-agarose were analyzed by 15% SDS-PAGE and silver staining.GST fusion proteins were further on a TSK-heparin column as described.

Results Differential Receptor Binding and Mitogenic Activities ofVEGF165 and VEGF121 for HWEC.

VEGF165 and VEGF121 differ in their ability to interact with VEGFreceptors expressed on HUVEC (Soker, et al., 3: Biol. Chem., 271,5761-5767 (1996), Gitay-Goren, et al., 3: Biol. Chem., 271, 5519-5523(1996)). VEGF 121 binds to KDR/Flk-1 to form a 240-kDa labeled complex(FIG. 1, lane 2), whereas VEGFI65 in addition to forming this sizecomplex, also forms a lower molecular mass complex of 165-175 kDa (FIG.1, lane 1). This isoform-specific receptor has been named the VEGF165receptor (VEGF165R). These differential receptor binding propertiessuggest that VEGF165 and VEGF121 might also have differential mitogenicactivities. Accordingly, the ability of the two VEGF isoforms tostimulate HUVEC proliferation was tested. VEGF165 was a more potentmitogen for HUVEC than was VEGF121 (˜FIG. 2). VEGF165 stimulatedhalf-maximal DNA synthesis at 1 ng/ml and maximal stimulation at 4 ng/mlresulting in an 8-fold increase over control. On the other hand, 2 ng/mlVEGF121 were required for half-maximal stimulation and 20 ng/ml formaximal stimulation resulting in a 4-fold increase in HUVECproliferation over control. Thus, twice as much VEGF121 compared withVEGF165 was needed to attain half-maximal stimulation, andVEGF121-induced proliferation was saturated at about one-half the levelinduced by VEGF165. Taken together, these results suggest that theremight be a correlation between the enhanced mitogenic activity ofVEGF165 for EC compared with VEGF121 and the ability of VEGF165 to bindto an additional receptor (VEGF165R) on HUVEC.

A Fusion Protein Containing the Exons 7- and 8-Encoded Domains Inhibitsthe Binding of 125I-VEGF165 to Receptors on HUVEC and 231 Cells.

Our previous studies indicated that the binding of VEGF165 to VEGF165Ris mediated by the 44 amino acids encoded by exon 7 (VEGF amino acids116-158) which is present in VEGF165 but absent in VEGF121 (Soker, etal., 3: Biol. Chem., 271, 5761-5767 (1996)). GST fusion proteinscontaining a peptide encoded by VEGF exon 7 or by VEGF exons 7 and 8were prepared. The 6 amino acids encoded by exon 8 which is C-terminalto exon 7 were included to facilitate the preparation of the fusionprotein but did not affect the results in any way (data not shown). Theexon 7 fusion protein binds directly to VEGF₁₆₅R on 231 cells (Soker, etal., J. BioL Chem., 271, 5761-5767 (1996)). It also binds directly toVEGF₁₆₅R on HUVEC but not to KDR/FLK-1 on HUVEC (FIG. 1, lane 3). Theability of the GST-VEGF₁₆₅ exons 7- and 8-encoded peptide (GST-Ex 7 & 8)to compete with ¹²⁵I-VEGF₁₆₅ binding to HUVEC, which express bothKDR/Flk-1 and VEGF₁₆₅R, to PAE-KDR cells which express only KDR/Flk-1(Waltenberger, et al., J. Biol. Chem., 269, 26988-26995 (1994)), and to231 cells which express only VEGF₁₆₅R (Soker, et al., J. BioL Chem.,271, 5761-5767 (1996)) was tested (FIG. 3). Increasing concentrations ofGST-Ex 7+8 markedly inhibited the binding of ¹²⁵I-VEGF₁₆₅ to HUVEC byabout 85-95% (FIG. 3A) and to 231 cells by 97-98% (FIG. 3B). However,the fusion protein did not inhibit the binding of ¹²⁵I-VEGF₁₆₅ toPAE-KDR cells which do not express any VEGF₁₆₅R (FIG. 3C). GST proteinalone even at concentrations of 20 μg/ml had no significant effect onthe binding of ¹²⁵I-VEGF₁₆₅ to any of the cell types. Taken together,these binding studies suggested that GST-EX 7+8 competes for¹²⁵I-VEGF₁₆₅ binding by interacting directly with VEGF₁₆₅R but not withKDR.

These binding experiments were extended to analyze the effects of GST-Ed7+8 on ¹²⁵I-VEGF binding to the individual VEGF receptor species bycross-linking (FIG. 4). Cross-linking of ¹²⁵I-VEGF₁₆₅ to 231 cellsresulted in the formation of labeled complexes with VEGF₁₆₅R (FIG. 4,lane 3). The formation of these complexes was markedly inhibited in thepresence of 15 ug/ml GST-Ex 7+8 (FIG. 4, lane 4). ¹²⁵I-VEGF₁₆₅cross-linking to HUVEC resulted in the formation of labeled complexes ofhigher molecular mass with KDR/Flk-1 and lower molecular mass complexeswith VEGF₁₆₅R (FIG. 4, lane 1). GST-Ex 7+8 markedly inhibited theformation of the VEGF₁₆₅-175-kDa labeled complexes containing VEGF₁₆₅R(FIG. 4, lane 2). On the other hand, the fusion protein did not inhibitcross-linking of ¹²⁵I-VEGF₁₆₅ to KDR/Flk-1 on the PAR/KDR cells (notshown). Taken together, since (i) VEGF₁₆₅ binds to KDR/Flk-1 via theamino acids encoded by exon 4 (40), (ii)) VEGF₁₆₅ binds to VEGF₁₆₅R viathe amino acids encoded by exon 7, and (iii) GST-Ex 7+8 binds toVEGF₁₆₅R but not to KDR (FIG. 1 and FIG. 8), these results suggestedthat by interfacing directly with the binding of ¹²⁵I-VEGF₁₆₅ toVEGF₁₆₅R, GST-Ex 7+8 also inhibits indirectly the binding of¹²⁵I-VEGF₁₆₅ to KDR/Flk-1.

Localization of the Core Inhibitory Region with the Exon 7-EncodedDomain.

The GST-Ex 7 fusion protein encompasses the entire 44 amino acid exon7-encoded domain. To determine whether a core inhibitory region exists,deletions were made at the N and C termini of exon 7, and the effects on¹²⁵I-VEGF₁₆₅ binding to HUVEC were measured (FIG. 5). In theseexperiments a fusion protein containing the exon 7-encoded domain plusthe cysteine residue at position of exon 8 was included to keep thenumber of cysteine residues in the VEGF portion of the fusion proteineven. The GST-Ex 7 fusion protein inhibited ¹²⁵I-VEGF₁₆₅ binding toHUVEC by 80% at 2 ug/ml fusion protein (FIG. 5). Inhibition of¹²⁵I-VEGF₁₆₅ binding to HUVEC and 231 cells was comparable to that ofGST-Ex 7+8 (data not shown). Deletion of the first 10 (GST-Ex 7d-(1-10))or 21 (GST-Ex 7d-(1-21)) N-terminal amino acids did not reduce theinhibitory activity of the fusion proteins. Actually, 1 ug/ml of GST-Ex7d-(1-21) had a greater inhibition activity than the same concentrationof GST-Ex 7 suggesting that there may be a region within exon 7 aminoacids 1-21 that interferes with the inhibitory activity. On the otherhand, deletion of the cysteine residue at position 22 in exon 7 (GST-Ex7d(1-22)) resulted in a complete loss of inhibitory activity. Deletionof the 15 C-terminal amino acids (GST-Ex 7 d-(30-44)) also resulted in acomplete loss of inhibitory activity (FIG. 5). These results indicatedthat the inhibitory core is found within amino acids 22-44 of exon 7.Moreover, it seems that the cysteine residue at position 22 in exon 7,which is Cys137 in VEGF, is crucial for maintaining a specific structurerequired for the inhibition.

GST-Ex 7+8 Inhibits VEGF [65-Induced Proliferation of HUVEC.

The inhibition of VEGF₁₆₅ binding to KDR/Flk-1 by the GST-Ex 7+8 fusionprotein as shown in FIG. 4 suggested that it might also be an inhibitorof VEGF₁₆₅ mitogenicity since KDR/Flk-1 mediates VEGF mitogenic activity(Waltenberger, et al., J. Biol. Chem., 269, 26988-26995 (1994)).Addition of 1-5 ng/ml VEGF₁₆₅ to HUVEC resulted in a 5.5-fold increasein the proliferation rate, peaking at 2.5 ng/ml (FIG. 6). When 15 ug/mlGST-Ex 7+8 was added in addition to VEGF₁₆₅. HUVEC proliferation wasreduced by about 60%. GST protein prepared in a similar way did notinhibit HUVEC proliferation even at 25 ug/ml indicating that theinhibitory effect was due solely to the presence of the exon 7+8-encodeddomain within the fusion protein. It was concluded that exon 7+8peptide-mediated inhibition of VEGF₁₆₅ binding to VEGF receptors onHUVEC correlates with the inhibition of HUVEC proliferation.

GST-Ex 7+8 Inhibits VEGF₁₂₁-Induced Proliferation of HUVEC.

GST-Ex 7+8 inhibits the level of VEGF₁₆₅-induced mitogenicity, about2-fold, to about the level of VEGF₁₂₁-induced mitogenicity (FIG. 7).GST-Ex 7+8, at 15 ug/ml, also inhibited VEGF₁₂₅-mediated HUVECproliferation, by about 2-fold. This was an unexpected resultconsidering that VEGF₁₂₁ does not contain exon 7. To understand betterthe nature of the VEGF₁₂₁ inhibition, the effect of GST-EX 7+8 on thebinding of ¹²⁵I-VEGF₁₂₁ to VEGF receptors was analyzed by cross-linkingstudies. Cross-linking of ¹²⁵I-VEGF₁₂₁ to HUVEC resulted in theformation of 240-kDa labeled complexes (FIG. 8, lane 1), which have beenshown to contain VEGF₁₂₁ and KDR/Flk-1 (Soker, et al., J. Biol. Chem.,271, 5761-5767 (1996), Gitay-Goren, et al., J. Biol. Chem., 271,5519-5523 (1996)). Formation of these complexes was significantlyinhibited by GST-Ex 7+8 at 15 ug/ml (FIG. 8, lane 2). It was concludedthat GST-Ex 7+8 inhibits VEGF₁₂₁-induced mitogenicity possibly byinhibiting its binding to KDR/Flk-1.

Discussion

The most abundant of the VEGF isoforms are VEGF₁₆₅ and VEFG₁₂₁. Animportant question in terms of understanding VEGF biology is whetherthese isoforms differ in their biochemical and biological properties. Todate, it has been demonstrated that VEGF₁₆₅ but not VEGF₁₂₁, binds tocell-surface HSPG (Houck, et al., Mol Endocrinol., 8, 1806-1814 (1991),Houck, et al., J. Biol. Chem., 247, 28031-28037 (1992), Park, et al.,Mol Biol. Cell, 4, 1317-1326 (1993)) and that VEGF₁₆₅ is a more potentEC mitogen than is VEGF₁₂₁ (Smith, et al., Gene (Amst.), 87, 31-40(1988)) (FIG. 2). In addition, we recently characterized a novel 130-kDaVEGF receptor found on the surface of HUVEC and tumor cells that isspecific in that it binds VEGF₁₆₅ but not VEGF₁₂₁ (Soker, et al., J.Biol. Chem., 271, 5761-5767 (1996)). VEGF₁₆₅ binds to this receptor,termed VEGF₁₆₅R, via the 44 amino acids encoded by exon 7, the exonwhich is present in VEGF₁₆₅ but not VEGF₁₂₁. In contrast KDR/Flk-1 andFlt-1 bind both VEGF₁₆₅ and VEGF₁₂₁ and do so via the VEGF exons 4 and3, respectively (Keyt, et al., J. Biol. Chem., 271, 5638-5646 (1996)).Our goal in the present study was to determine whether exon 7 modulatedVEGF₁₆₅ activity, in particular mitogenicity for HUVEC, and by whatmechanism. To do so, we developed a strategy of inhibiting the bindingof VEGF₁₆₅ to VEGF₁₆₅R using a GST fusion protein containing the exon7-encoded domain and examining any subsequent effects on HUVECproliferation. Cross-linking experiments demonstrated, as expected, thatthe exon 7 fusion protein could bind to VEGF₁₆₅R but not to KDR/Flk-1.The exon 7 fusion protein was found to be a potent inhibitor of¹²⁵I-VEGF₁₆₅ binding to 231 cells which express VEGF₁₆₅R alone, by 98%and to HUVEC which express both KDR/Flk-1 and VEGF₁₆₅R, by 85-95%. Itdid not, however, inhibit at all the binding of ¹²⁵I-VEGF₁₆₅ to PAE-KDRcells which express KDR/Flk-1 but not VEGF₁₆₅R. GST protein alone didnot inhibit binding to any of the cell types demonstrating that theinhibition was due solely to the exon 7 portion of the fusion protein.Cross-linking analysis, which demonstrated the formation of specific¹²⁵I-VEGF₁₆₅ receptor complexes, confirmed that GST-Ex 7+8 markedlyinhibited the binding of ¹²⁵I-VEGF₁₆₅ to VEGF₁₆₅R on HUVEC and 231cells. Taken together, these results indicate that the exon 7 fusionprotein interacts directly with VEGF₁₆₅R and can act as a competitiveinhibitor of binding of ¹²⁵I-VEGF₁₆₅ to this receptor.

The GST-Ex 7+8 fusion protein inhibited VEGF₁₆₅-induced proliferation ofHUVEC by about 60%, to a level equivalent to that induced by VEGF₁₂₁suggesting that activation of the KDR/Flk-1 tyrosine kinase receptor wassomehow being adversely affected. Indeed, cross-linking analysis showedthat the fusion protein not only inhibited cross-linking of ¹²⁵I-VEGF₁₆₅to VEGF₁₆₅R but to KDR/Flk-1 as well. This result was unexpected sinceour cross-linking studies show that the exon 7 fusion protein does notbind directly to KDR/Flk-1 consistent with the previous demonstrationthat VEGF₁₆₅ interacts with KDR/Flk-1 via its exon 4-encoded domain(Keyt, et al., J. Biol. Chem., 271, 5519-5523 (1996)). Thus it appearsthat the binding of ¹²⁵I-VEGF₁₆₅ to VEGF₁₆₅R via the exon 7-encodeddomain modulates indirect interaction of the growth factor withKDR/Flk-1. A possible mechanism for this inhibitory effect of GST-Ex 7+8on HUVEC proliferation is that KDR/Flk-1 and VEGF₁₆₅R are co-localizedin close proximity on the cell surface. In this model, a VEGF₁₆₅ dimerinteracts simultaneously with KDR/Flk-1 via the exon 4 domain and withVEGF₁₆₅R via the exon 7 domain, generating a three-component complex.The GST-Ex 7+8 fusion protein by competing directly with the binding ofVEGF₁₆₅ to VEGF₁₆₅R impairs indirectly the ability of VEGF₁₆₅ to bind tothe signaling receptor, KDR/Flk-1. Thus, an efficient binding of VEGF₁₆₅to KDR/Flk-1 might be dependent in part on successful interaction withVEGF₁₆₅R. An alternative possibility is that the exon 7-encoded domaincontains a heparin-binding domain (Soker, et al., J. Biol Chem., 271,5761-5767 (1996)) and that an excess of GST-Ex 7+8 prevents VEGF₁₆₅ frombinding to cell-surface HSPGs that are required for efficient binding ofVEGF₁₆₅ to its receptors (Gitay-Goren, et al., J Biol. Chem., 287,6003-6096 (1992)).

Surprisingly, GST-Ex 7+8 also inhibited the mitogenic activity ofVEGF₁₂₁ for HUVEC, by about 50%, even though VEGF₁₂₁ does not bind toVEGF₁₆₅R (Soker, et al., J. Biol. Chem., 271, 5761-5767 (1996)). Apossible explanation is that VEGF₁₆₅R and KDR/Flk-1 are in proximity onthe cell surface and that excess GST-Ex 7+8 bound to VEGF₁₆₅R stericallyinhibits access of VEGF₁₂₁ to KDR/Flk-1. Cross-linking analysis didindeed show diminished binding of ¹²⁵I-VEGF₁₂₁ to KDR/Flk-1 in thepresence of GST-Ex 7+8 which does not bind directly to KDR/Flk-1,suggesting an indirect effect of the fusion protein on the binding ofVEGF₁₂₁ to KDR/Flk-1.

GST-Ex 7+8 also inhibits VEGF₁₆₅ binding to 231 breast cancer cells,which express VEGF₁₆₅R and not KDR/Flk-1.

The coordinate binding of VEGF₁₆₅ to a higher and to a lower affinityreceptor (KDR/Flk-1 and VEGF₁₆₅R, respectively) on HUVEC (Soker, et al.,J Biol. Chem., 271, 5761-5767 (1996)) and the inhibitory effects ofGST-Ex 7+8 fusion protein on the binding of VEGF₁₆₅ to these tworeceptors suggest that there is a dual receptor system at work inmediating VEGF₁₆₅ activity. Several other growth factors have been shownto bind to high and low affinity receptors. Transforming growth factor-βgenerates a complex with three receptors; two of the, receptors I andII, are the signaling receptors, whereas transforming growth factor-Breceptor III/betaglycan is a lower affinity accessory binding molecule(Lopez-Casillas, et al., Cell, 47, 785-796 (1991)). The low affinityreceptor for the nerve factor family, p75, is part of a complex with thesignaling TRK receptors (Barbacid, M., Curr. Opin. Cell Biol., 7,148-155 (1995)). A different type of dual receptor recognition is thebinding of bFGF to cell-surface HSPGs and its signaling receptors(Yayon, et al., Cell, 64, 841-848 (1991), Klagsbrun, et al., Cell, 67,229-231 (1991)). It has been suggested that binding of bFGF to its lowaffinity receptors (HSPGS) may induce conformational changes in bFGF sothat the HSPG-bound bFGF could be efficiently presented to its highaffinity, signaling receptors (Yayon, et al., Cell, 64, 841-848 (1991),Klagsbrun, et al., Cell, 67, 229-231 (1991)). Thus, the binding ofVEGF₁₆₅ to both VEGF₁₆₅R and KDR/Flk-1 appears to be part of a generalmechanism wherein two different types of receptors are used to modulategrowth factor activity.

Receptor binding studies were used to identify an inhibitory core withinthe 44 amino acids encoded by exon 7. Deletions were made in both theN-terminal and C-terminal regions of exon 7, and the inhibitory activitywas localized to the 23-amino acid C-terminal portion of exon 7 (aminoacids 22-44). Of these 23 amino acids, 5 are cysteine residues. The highproportion of cysteine residues suggests that this domain has a definedthree-dimensional structure required for efficient binding to VEGF₁₆₅R.The cysteine residue at position 22 of the exon 7 domain is critical forinhibitory activity, probably for maintenance of a necessarythree-dimensional structure. A study that examined the role of cysteineresidues at different positions in VEGF₁₆₅ showed that a substitution ofCys146, which lies within the core inhibitory domain of exon 7 (atposition 31 in exon 7), by a serine residue resulted in a 60% reductionin VEGF₁₆₅ permeability activity and a total loss of EC mitogenicity(Claffey, et al., Biochim, Biophys. Acta., 1246, 1-9 (1995)). The Cys146mutation had no effect on the dimerization of VEGF (Claffey, et al.,Biochim, Biophys. Acta., 1246, 1-9 (1995)). Thus, it appears that thiscysteine residue is not involved in the formation of interdisulfidebounds between two VEGF monomers but might rather involve intradisulfidebonding within the monomer. These results support our hypothesis that athree-dimensional structure stabilized by cysteine residues exists inthe C-terminal half of exon 7 that contributes to VEGF₁₆₅ biologicalactivity, such as interaction with VEGF165R. Interestingly, a fusionprotein corresponding to a deletion of the N-terminal 21 amino acidresidues encoded by exon 7 was a more potent inhibitor than the intactexon 7-encoded peptide. It may be that the N-terminal portion results inenhanced binding to VEGF₁₆₅R and yields a better competitor of VEGF₁₆₅.

The references cited throughout the specification are incorporatedherein by reference.

The present invention has been described with reference to specificembodiments. However, this application is intended to cover thosechanges and substitutions which may be made by those skilled in the artwithout departing from the spirit and the scope of the appended claims.

1. A pharmaceutical composition comprising an isolated polypeptidehaving a portion of SEQ ID NO:1 having VEGF antagonist activity, and apharmaceutically acceptable carrier.
 2. A pharmaceutical compositioncomprising an isolated polypeptide comprising SEQ ID NO:2 or a portionthereof having VEGF antagonist activity, and a pharmaceuticallyacceptable carrier.
 3. A pharmaceutical composition comprising anisolated polypeptide comprising a peptide having the structure of thefollowing formula (I): (SEQ ID NO: 3) (X₁-(CSCKNTDSRCKARQLELNERT)X₂ I

wherein X₁ is H, or any portion of amino acids 2-21 of SEQ ID NO:1, andX₂ is H or C, CR, RC, or CRC, and analogs thereof, and apharmaceutically acceptable carrier.
 4. The pharmaceutical compositionof claim 1, wherein the carrier is acceptable for topical application tothe skin.
 5. The pharmaceutical composition of claim 1, wherein thecarrier is acceptable for application to the eye.
 6. The pharmaceuticalcomposition of claim 2, wherein the carrier is acceptable for topicalapplication to the skin.
 7. The pharmaceutical composition of claim 2,wherein the carrier is acceptable for application to the eye.
 8. Thepharmaceutical composition of claim 3, wherein the carrier is acceptablefor topical application to the skin.
 9. The pharmaceutical compositionof claim 3, wherein the carrier is acceptable for application to theeye.